Construction Of BCGΔWag22 Mutant In Bcg And Latent Infection Model In BALB/c

Mycobacterium tuberculosis is the main pathogen of tuberculosis. Tuberculosis is a worldwide disease which is caused great harm in the spiritual and material. When completing the Mycobacterium tuberculosis genome sequencing, we found a region which is have 10% of the whole genome sequence of MTB, and this region is so called PE/PPE family. According to the sequence of C terminal, The PE family can be divided into two subfamily-pe and pe-pgrs; The PPE family can be divided into four subfamily---ppe-mptr(major polymor phictandem repeats), ppe-svp(with a GxxSVPxxW motif), ppe-ppw(with a PxxPxxW motif) and ppe(with no distinctive features). In our stady, the objective gene Wag22 belongs to pe-prgs subfamily, this subfamily is characterized by high GC content of the C sequences(more than 60%) and contains much GC repeats, it increases the difficulty of ampling of the target genes in vitro.Only a small amount of literature on Wag22 has been reported, pointed out that, Wag22 can only express when infect the host, and its expression product Wag22 protein have fibronectin activity, it may play a role in the early of infection with Mycobacterium tuberculosis. Our stady is based on BCG, we will knockout Wag22, comparing the impaction when the two bacterias interact with the RAW264.7 macrophage of BALB/c rat, to explore the role of Wag22 in the infection of the host. And establishing an animal model of latent infection with BCG and deletion strain.(1) construction of the mutant of BCGWe construct a mycobacterium phage, which contains the up and downstream homologous arms of Wag22 and the hyg and sacB selection genes. When the phage infects BCG, it will inject them into BCG, then homologous recombination happens. We can screen the positive clones in the Hyg resresistance plate,and identify the right mutant by PCR.(2) Phenotype of mutant BCGΔ Wag22We tested the colony morphology, growth curve, cell toxicity, anti phagocytic and survival ability of the mutant and BCG, finding that after kncokout Wag22 the colony morphology and growth curve have no significant difference. About the ability of resistance to phagocytosis and viability, the mutant is more easily be swallowed in macrophage but the survival rate in macrophage has no difference. And the mutant strain was more toxic to RAW264.7 cells.These changes maybe represent the mutant strains of BCG is more easier access to the host and long-term in host, to stimulate the host to produce long-term immunity.(3) The expression of Wag22 protein in vitroDue to the protein Wag22 only expresses when infected the host, we expressed the target protein in vitro. And the prokaryotic expression vector with target gene was constructed successfully. The expression form of target protein is spheroidins. Because the protein concentration we obtained is too low, we can’t carry on the next experiment. We will try other methods to obtain the target protein which is meeting the requirements.(4) Construct an animal model of latent infectionWe established the latent infection model using the ΔWag22 and BCG, we use 6-8 weeks female BALB/c mouse, 5×105 CFU/200μL bacterias were injected into the mice by tail vein injection, the experiment lasted for two weeks, in the middel, we will inject 10 mg dexamethasone each mouse, then to observe the related pathological changes of every organ, we found that the livers and spleens showed obvious pathological changes. But the hearts, lungs, and kidneys showed nothing.Unfortunately, we did not find the granuloma. we also detected the cytokines IL-6, INF-γ, IL-2 and TNF-α, we found the mutant can cause more higher IL-6, INF-γ and TNF-α, it represents that the mutant ΔWag22 can induce stronger immune response than BCG.